Establishment and application of human immortalized b lymphocyte cell line group

ABSTRACT

The present application provides a group of human immortalized B lymphocyte cell lines and use thereof, and specifically provides a combination of four closely related immortalized lymphocyte cell lines. The combination can be used as a standard product for accuracy evaluation of a detection platform. When the four closely related immortalized lymphocyte cell lines are used as standard products for epigenome, transcriptome, proteome, and metabolome, an intrinsic magnitude difference gradient can be formed to evaluate the sensitivity of histological detection.

TECHNICAL FIELD

The present invention relates to the field of biotechnology, inparticular, to the establishment and application of a group of humanimmortalized B lymphocyte lines.

BACKGROUND

Immortalized cell line refers to a cell line that can proliferateindefinitely and continue to survive through passage after successfulprimary culture. Using Epstein-Barr virus (EBV) to transform peripheralblood B lymphocytes to establish an immortalized cell line is the mostcommonly used method for cell immortalization. Epstein-Barr virus is aherpes virus that is B-lymphophilic, belonging to the herpes virusfamily lymphotropic virus, and is a linear double-stranded DNA virus.After EBV infection of B lymphocytes, it causes the activation of Blymphocytes, the cells proliferate endlessly, and transform into animmortal lymphoblastoid cell line.

Since immortalized cell lines can provide cell models for variousgenetic diseases, immunology, and cell biology research under subcultureconditions, it has been widely used in the preservation of geneticmaterial and is used by the HapMap International Project for thepreservation of genetic material.

At the same time, a large number of immortalized cell lines have beenexpanded. The National Institute of Standards and Technology (NIST) hasused immortalized cell lines to prepare DNA in large quantities and madeRM8398, a reference substance for whole genome DNA.

However, with the increase of new technology platforms forhigh-throughput omics testing, the requirements for the accuracy ofstandard products and the coverage of omics testing technology platformshave gradually increased. For example, DNA sequencing analysis requiresmore consideration of haplotype results. It is also necessary to combineparents' sequencing data to obtain de-novo mutation information, so thata set of reference substances for family DNA is required. At the sametime, the reliability and reproducibility of high-throughput detectiontechnology platforms such as epigenome, transcriptome, and proteome alsourgently need to be systematically evaluated. A set of different samplesis needed to evaluate the relative magnitude of expression. Currently,there is no family-designed immortalized cell line used as a standardproduct for a high-throughput omics testing technology platform.

Therefore, there is an urgent need to develop a standard product thatcan judge the accuracy of the sequencing platform.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a standard productthat can judge the accuracy of the sequencing platform.

In a first aspect of the present invention, it provides a cell line forconstructing a standard product for judging the accuracy of a detectionplatform, the cell line is selected from the group consisting of: humanimmortalized B lymphocyte line Fudan_D5, human immortalized B Lymphocyteline Fudan_D6, human immortalized B lymphocyte line Fudan_F7, and/orhuman immortalized B lymphocyte line Fudan_M8; and, the deposit numberof human immortalized B lymphocyte line Fudan_D5 is CCTCC NO: C2017238,the deposit number of the human immortalized B lymphocyte line Fudan_D6is CCTCC NO: C2017253, the deposit number of the human immortalized Blymphocyte line Fudan_F7 is CCTCC NO: C2017254, and the deposit numberof the human immortalized B lymphocyte line Fudan_M8 is CCTCC NO:C2017255.

In another preferred embodiment, the cell line comprises humanimmortalized B lymphocyte line Fudan_D5, human immortalized B lymphocyteline Fudan_D6, human immortalized B lymphocyte line Fudan_F7 and humanimmortalized B lymphocyte line Fudan_M8.

In a second aspect of the present invention, it provides a standardproduct, which is extracted from the cell line according to the firstaspect of the present invention.

In another preferred embodiment, the standard product comprises a set ofstandards, and each standard is derived from the cell line according tothe first aspect of the present invention, respectively.

In another preferred embodiment, the standard is selected from the groupconsisting of: a nucleic acid standard, a protein standard, a metabolitestandard, and a combination thereof.

In another preferred embodiment, the standard product is of the sametype.

In another preferred embodiment, the nucleic acid standard comprises aDNA standard and/or a RNA standard.

In another preferred embodiment, the standard comprises a firststandard, a second standard, a third standard, and a fourth standard;wherein, the first standard is extracted from a human immortalized Blymphocyte line Fudan_D5, the second standard is extracted from humanimmortalized B lymphocyte line Fudan_D6, the third standard is extractedfrom human immortalized B lymphocyte line Fudan_F7, and the fourthstandard is extracted from human immortalized B lymphocyte lineFudan_M8.

In a third aspect of the present invention, it provides a use of thestandard product according to the second aspect of the present inventionto prepare a reagent and/or kit for judging the accuracy of a detectionplatform.

In another preferred embodiment, the detection platform is selected fromthe group consisting of a sequencing platform, a chip detectionplatform, a metabolite detection platform, a methylation detectionplatform, a transcriptome detection platform, a proteome detectionplatform, and a combination thereof.

In another preferred embodiment, the sequencing platform is selectedfrom the group consisting of a high-throughput DNA detection platform, ahigh-throughput RNA detection platform, and a combination thereof.

In another preferred embodiment, the chip detection platform comprises agene chip detection platform and/or a protein chip detection platform.

In a fourth aspect of the present invention, it provides a method forjudging the accuracy of a detection platform to be tested, comprisingthe steps:

(a) providing a standard product according to the second aspect of thepresent invention and a platform to be tested;

(b) constructing a library for the standard product, thereby obtaining asequencing library;

(c) sequencing the sequencing library of step (b) using the platform tobe tested, thereby obtaining a sequencing result;

(d) comparing the sequencing result with the standard data (thresholdvalue) of the standard sequence corresponding to the standard product,thereby obtaining the performance confirmation parameters of the method,including precision, accuracy, sensitivity, specificity, and/ordetectable range, thereby evaluating and verifying the accuracy of theplatform to be tested.

In another preferred embodiment, in step (d), comparing the sequencingresult with the standard data (threshold value) of the standard sequencecorresponding to the standard product, thereby obtaining the matchingvalue of the sequencing result and the standard data of the standardsequence, if the matching value is ≥99% (preferably ≥99.5%, morepreferably, 99.974-99.980%), indicating that the sequencing platform isaccurate or qualified; if the matching value is <98%, indicating thatthe sequencing platform is inaccurate or unqualified.

In another preferred embodiment, the method further comprises: (e)analyzing the STR of the standard product, and comparing the obtainedSTR analysis result with the STR analysis result corresponding to thestandard product.

In another preferred embodiment, based on the comparison result of step(e), thereby obtaining the matching value between the STR analysisresult and the STR analysis result of the standard product, if thematching value is ≥98% (preferably, ≥99%, more preferably, ≥99.5%),indicating that the sequencing platform is accurate or qualified, if thematching value is less than 96%, indicating that the sequencing platformis inaccurate or unqualified.

In another preferred embodiment, the method further comprises: (f)analyzing the Mendelian error rate of the standard product, andcomparing the obtained Mendelian error rate results with the Mendelianerror rate results corresponding to the standard product.

In another preferred embodiment, based on the comparison result of step(f), obtaining the matching value between the result of the Mendelianerror rate and the Mendelian error rate of the standard product, if thematching value is ≥95% (preferably, ≥98%, more preferably, ≥99%),indicating that the sequencing platform is accurate or qualified, if thematching value is less than 95%, indicating that the sequencing platformis inaccurate or unqualified.

In another preferred embodiment, the method further comprises: (g)analyzing the methylation of the standard product, and comparing theobtained methylation analysis result with the methylation analysisresult corresponding to the standard product.

In another preferred embodiment, based on the comparison result of step(g), thereby obtaining the matching value of the methylation analysisresult and the methylation analysis result corresponding to the standardproduct, if the matching value is ≥98% (preferably, ≥99%, morepreferably, ≥99.5%), indicating that the sequencing platform is accurateor qualified, and if the matching value is less than 96%, indicatingthat the sequencing platform is inaccurate or unqualified.

In another preferred embodiment, the platform to be tested is sequencedby a high-throughput sequencer selected from the group consisting of:Illumina HiSeq XTEN, Illumina HiSeq 2500, Illumina HiSeq 2000, IlluminaNovaSeq, MiSeq, Ion Torrent, BGISeq-500, PacBio, BioNano, Nanopore and acombination thereof.

In a fifth aspect of the present invention, it provides a kitcomprising:

(a) a first container, and a first standard located in the firstcontainer, the first standard is extracted from a human immortalized Blymphocyte strain Fudan_D5;

(b) a second container, and a second standard located in the secondcontainer, the second standard is extracted from a human immortalized Blymphocyte strain Fudan_D6;

(c) a third container, and a third standard located in the thirdcontainer, the third standard is extracted from a human immortalized Blymphocyte strain Fudan_F7; and

(d) a fourth container, and a fourth standard located in the fourthcontainer, the fourth standard is extracted from a human immortalized Blymphocyte strain Fudan_M8.

In another preferred embodiment, the kit further comprises instructions,which record instructions for using the first standard, the secondstandard, the third standard, and the fourth standard for judging theaccuracy of the detection platform.

In another preferred embodiment, the first container, the secondcontainer, the third container and the fourth container are differentcontainers.

In another preferred embodiment, the standard is selected from the groupconsisting of: a nucleic acid standard, a protein standard, a metabolitestandard, and a combination thereof.

In another preferred embodiment, the nucleic acid standard comprises aDNA standard and/or an RNA standard.

It should be understood that, within the scope of the present invention,the technical features specifically described above and below (such asthe Examples) can be combined with each other, thereby constituting anew or preferred technical solution which needs not be described one byone.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the morphology of the immortalized B lymphocyte line (phasecontrast microscope, 40×).

FIG. 2 shows the results of the STR method to identify the geneticrelationship between the four cell lines and the original blood sampleDNA.

FIG. 3 shows the cluster diagram of the methylation level of 4immortalized cell lines detected by the Illumina850K methylation chip.

FIG. 4 shows the cluster diagram of the mRNA expression levels of 4immortalized cell lines detected by transcriptome sequencing.

DETAILED DESCRIPTION OF INVENTION

After extensive and intensive research and extensive screening, theinventors have unexpectedly found that 4 immortalized lymphocyte lineswith genetic relationships can be combined as standard products forjudging the accuracy of the detection platform, and when 4 immortalizedlymphocyte lines with genetic relationship are used as standard productsof epigenome and transcriptome, it can form an intrinsic magnitudedifference gradient for evaluating the sensitivity of omics detection.On this basis, the inventors have completed the present invention.

Construction of Cell Lines for Standard Products Used to Identify theAccuracy of DNA Sequencing

The present invention provides a cell line for constructing a standardfor identifying the accuracy of DNA sequencing. The cell line isselected from the group consisting of: a human immortalized B lymphocytecell lineFudan_D5, a human immortalized B lymphocyte cell lineFudan_D6,human immortalized B-lymphocyte cell lineFudan_F7, human immortalizedB-lymphocyte cell lineFudan_M8, and a combination thereof; and thedeposit number of human immortalized B-lymphocyte cell lineFudan_D5 isCCTCC NO: C2017238, the deposit number of human immortalized Blymphocyte cell lineFudan_D6 is CCTCC NO: C2017253, the deposit numberof the human immortalized B lymphocyte cell lineFudan_F7 is CCTCC NO:C2017254, and the deposit number of the human immortalized B lymphocytecell lineFudan_M8 is CCTCC NO: C2017255.

The immortalized B lymphocyte lines Fudan_D5, Fudan_D6, Fudan_F7,Fudan_M8 of the present invention can be used for large-scalepreparation of standard products of DNA, RNA, protein, and metabolites,and can also be used for studying genetic relationships.

The present invention adopts the method of transfecting human Blymphocytes with EBV to obtain a set of immortalized cell lines that canbe passaged stably and retain the genetic characteristics of theoriginal blood sample. This group of immortalized B lymphocyte lines isfrom the family of identical twins, Fudan_D5 and Fudan_D6 are fromidentical twins, Fudan_F7 is from the father, and Fudan_M8 is from themother.

The group of cell lines of the present invention has the followingcharacteristics:

1) the genetic background is clear and the biological characteristicsare stable. The immortalized cell line identified by the STR method isconsistent with the original blood sample, the sequence characteristicsof the whole genome thereof are more than 99.97% consistent with theoriginal blood sample, and it remains stable after multiple passages.

2) the family design of identical twins is suitable for studying thegenetic relationship between offspring and parents and the correspondingomics characteristics.

Standard Product

The present invention provides a standard product, which is extractedfrom the cell line used for judging the accuracy of a sequencingplatform of the present invention.

In another preferred embodiment, the standard product comprises a set ofstandards, and each standard is from the cell line according to thefirst aspect of the present invention.

In another preferred embodiment, the standard is selected from the groupconsisting of: a nucleic acid standard, a protein standard, a metabolitestandard, and a combination thereof.

In another preferred embodiment, the standard comprises a firststandard, a second standard, a third standard, and a fourth standard,the first standard is extracted from a human immortalized B lymphocytecell line Fudan_D5, the second standard is extracted from a humanimmortalized B lymphocyte cell line Fudan_D6, the third standard isextracted from a human immortalized B lymphocyte cell line Fudan_F7, andthe fourth standard is extracted from a human immortalized B lymphocytecell line Fudan_M8.

In another preferred embodiment, the standard product is of the sametype.

In another preferred embodiment, the nucleic acid standard includes aDNA standard and/or an RNA standard.

In another preferred embodiment, the DNA standard includes a first DNAstandard, a second DNA standard, a third DNA standard, and a fourth DNAstandard, and the first DNA standard is extracted from a humanimmortalized B lymphocyte cell line Fudan_D5, the second DNA standard isextracted from a human immortalized B lymphocyte cell line Fudan_D6, thethird DNA standard is extracted from a human immortalized B lymphocytecell line Fudan_F7, and the fourth DNA standard is extracted from ahuman immortalized B lymphocyte cell line Fudan_M8.

In another preferred embodiment, the RNA standard includes a first RNAstandard, a second RNA standard, a third RNA standard, and a fourth RNAstandard, and the first RNA standard is extracted from a humanimmortalized B lymphocyte cell line Fudan_D5, the second RNA standard isextracted from a human immortalized B lymphocyte cell line Fudan_D6, thethird RNA standard is extracted from a human immortalized B lymphocytecell line Fudan_F7, and the fourth RNA standard is extracted from ahuman immortalized B lymphocyte cell line Fudan_M8.

Methods for Judging the Accuracy of Sequencing Platforms

The present invention provides a method for judging the accuracy of asequencing platform, comprising the steps:

(a) providing a standard product according to the second aspect of thepresent invention and a platform to be tested;

(b) constructing a library for the standard product to obtain asequencing library;

(c) sequencing the sequencing library of step (b) using the platform tobe tested, thereby obtaining a sequencing result;

(d) comparing the sequencing result with the standard data (thresholdvalue) of the standard sequence corresponding to the standard product,thereby obtaining the performance confirmation parameters of the method,including precision, accuracy, sensitivity, specificity, and/ordetectable range, thereby evaluating and verifying the accuracy of theplatform to be tested.

In another preferred embodiment, in step (d), compare the sequencingresult with the standard data (threshold value) of the standard sequencecorresponding to the standard product, thereby obtaining the matchingvalue between the sequencing result and the standard data of thestandard sequence, if the matching value is ≥99% (preferably, ≥99.5%,more preferably, 99.974-99.980%), indicating that the sequencingplatform is accurate or qualified; if the matching value is less than98%, indicating that the sequencing platform is inaccurate orunqualified.

In another preferred embodiment, the method further includes: (e)analyzing the STR of the standard product, and comparing the obtainedSTR analysis result with the STR analysis result corresponding to thestandard product.

In another preferred embodiment, based on the comparison result of step(e), thereby obtaining the matching value between the STR analysisresult and the STR analysis result of the standard product, if thematching value is ≥98% (preferably, ≥99%, more preferably, ≥99.5%),indicating that the sequencing platform is accurate or qualified, if thematching value is less than 96%, indicating that the sequencing platformis inaccurate or unqualified.

In another preferred embodiment, the method further includes: (f)analyzing the Mendelian error rate of the standard product, andcomparing the obtained Mendelian error rate result with the Mendelianerror rate result corresponding to the standard product.

In another preferred embodiment, based on the comparison result of step(f), thereby obtaining the matching value between the result of theMendelian error rate and the Mendelian error rate of the standardproduct, if the matching value is ≥95% (preferably, ≥98%, morepreferably, ≥99%), indicating that the sequencing platform is accurateor qualified, if the matching value is less than 95%, indicating thatthe sequencing platform is inaccurate or unqualified.

In another preferred embodiment, the method further comprises: (g)analyzing the methylation of the standard product, and comparing theobtained methylation analysis result with the methylation analysisresult corresponding to the standard product.

In another preferred embodiment, based on the comparison result of step(g), thereby obtaining the matching value between the methylationanalysis result and the methylation analysis result corresponding to thestandard product, if the matching value is ≥98% (preferably, ≥99%, morepreferably, ≥99.5%), indicating that the sequencing platform is accurateor qualified, and if the matching value is less than 96%, indicatingthat the sequencing platform is inaccurate or unqualified.

In the present invention, the platform to be tested is not particularlylimited, and may be self-built by the laboratory or purchasedcommercially. In a preferred embodiment, the platform to be tested inthe present invention is self-built by the laboratory.

The advantages of the present invention mainly include:

(1) The present invention constructs a group of family immortalizedlymphocyte lines for the first time as a standard product for evaluatingthe accuracy of the sequencing platform. This cell line can be passagedstably and retain the genetic characteristics of the original bloodsample.

(2) In the present invention, when four immortalized lymphocyte lineswith genetic relationships are used as reference substances forepigenome, transcriptome, proteome, and metabolome for the first time,an intrinsic magnitude difference gradient can be formed for theevaluation of the sensitivity of omics detection.

(3) The use of the standard product in the present invention is toperform Validation on a new detection platform, laboratory, and method,and obtain a Performance Metrics, including precision, accuracy,sensitivity, specificity, detectable range, etc.

(4) The present invention provides more information for metabolitedetection platform, methylation detection platform, transcriptomedetection platform, and proteome detection platform.

(5) The standard product of the present invention can finally determinethe threshold through data analysis on a large number of platforms anddiscovering the distribution law.

The present invention will be further explained below in conjunctionwith specific embodiments. It should be understood that theseembodiments are only used to illustrate the present invention and not tolimit the scope of the present invention. The experimental methodswithout specific conditions in the following examples are usually basedon conventional conditions, such as the conditions described in Sambrooket al., Molecular Cloning: Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989), or according to the conditions suggestedby the manufacturer. Unless otherwise specified, percentages and partsare percentages by weight and parts by weight.

Unless otherwise specified, the materials and reagents used in theexamples are all commercially available products.

Example 1 Establishment of Human Immortalized B Lymphocytes

The study was approved by the Ethics Committee of the School of LifeSciences, Fudan University (Lunyan Batch No. 279), and all of the 4healthy volunteers signed an informed consent form. 15 mL of venousblood (EDTA anticoagulation) was collected from 4 volunteers for theestablishment of B lymphocyte immortalization cell lines. The fourhealthy volunteers were born in Taizhou, Jiangsu, and belonging to agroup of identical twins family.

(1) PBMC Separation

Transferring 15 mL of EDTA anticoagulated whole blood to a centrifugetube, centrifuged at 2000 rpm for 5 minutes, and separating the plasmaand blood cell layer.

The blood cells after centrifugation were diluted to 30 ml with PBS,mixed well, and slowly added to the top of the Ficoll separationsolution. Centrifuged at 2000 rpm for 20 minutes. Using a pipette tofirst remove the upper liquid, and then carefully aspirate the whiteblood cells, and transfer it to a new centrifuge tube.

Adding PBS to 30 ml, mixed upside down, centrifuged, and repeated threetimes.

(2) Naïve B Cell Sorting

Preparing Sorting buffer (PBS without calcium and magnesium ions+2%FBS+1 mM EDTA). The Naïve B cell sorting test was carried out accordingto the operation steps of EasySep™ Human Naïve B Cell Enrichment Kit(STEMCELL™, Catalog #19254).

(3) EBV Infection

Washing the sorted Naïve B cells with PBS once, counting them, andresuspending the cells with complete medium containing virus (the amountof EBV virus was 10-20% of the total volume) according to the countresults, the cell number was 1×105/200 μL/well (48-well plate).Centrifugation and infection were performed at 2000 rpm for 1 hour, andthen it was placed in an incubator for culture.

As shown in FIG. 1, the successfully transfected cells grow insuspension in clusters, in line with the typical characteristics of EBVtransfected B lymphocytes.

Example 2 Identification of the Source of the Cell Line Using STR Method

Short tandem repeat (STR), also known as microsatellite DNA, is a classof DNA sequence formed by tandem repeats with 2-6 base pairs as the coreunit. Since the number of core unit repeats is highly variable andabundant among individuals, it constitutes the genetic polymorphism ofthe STR locus. It is the second-generation genetic marker afterrestricted fragment length polymorphism (RFLP) and can be used forfamily identification of identical twins.

(1) Purification of Blood Sample DNA

Using the original blood sample of the volunteers before theestablishment of the line, adding 20 μL proteinase K and 50 μLanticoagulant blood into a 1.5 mL EP tube, making up the total volume to220 μL with PBS, and proceeding to step 2.

Adding 200 μL Buffer AL, mixed well, and placed in a 56° C. water bathfor 10 min.

Adding 200 μL of ethanol (96-100%) to the above sample and mixed well.

Transferring the above liquid to the purification column (DNeasy Minispin column) and placing it in the 2 ml collection tube provided by thekit. Centrifuged at 6000 g (or 8000 rpm) for 1 min. Discarding the wasteliquid and collection tube.

Putting the purification column into a new 2 ml collection tube, adding500 μL Buffer AW1, and centrifuged at 6000 g (or 8000 rpm) for 1 min.Discarding the waste liquid and collection tube.

Putting the purification column in a new 2 ml collection tube, adding500 μL Buffer AW2, and centrifuged at 20000 g (or 14000 rpm) for 3 min.Discarding the waste liquid and collection tube.

Putting the purification column in a new 1.5 ml or 2 ml centrifuge tube,adding 200 μL of Buffer AE, dropping it directly on the white membranein the middle, placed at room temperature for 1 min, and centrifuged at6000 g (or 8000 rpm) for 1 min to elute DNA.

The eluted DNA was quantified with Nanodrop, and the sample was dilutedto a concentration of 10 ng/μL, and 10 μL was taken for STR detection.

(2) Purification of Cell Line DNA

The Fudan_D5, Fudan_D6, Fudan_F7, Fudan_M8 cells were centrifuged at 300g for 5 min, and resuspended in 200 μL PBS solution. 20 μL proteinase Kwas added.

Adding 200 μL Buffer AL. Mixed well and placed in a 56° C. water bathfor 10 min.

Adding 200 μL of ethanol (96-100%) to the above sample, and make surethat the ethanol and sample were thoroughly mixed.

Transferring the liquid from the above steps to the purification column(DNeasy Mini spin column) and placed in the 2 ml collection tubeprovided in the kit. Centrifuged at 6000 g (or 8000 rpm) for 1 min.Discarding the waste liquid and collection tube.

Putting the purification column into a new 2 ml collection tube, adding500 μL Buffer AW1, and centrifuged at 6000 g (or 8000 rpm) for 1 min.Discarding the waste liquid and collection tube.

Putting the purification column into a new 2 ml collection tube, adding500 μL Buffer AW2, and centrifuged at 20000 g (or 14000 rpm) for 3 min.Discarding the waste liquid and collection tube. Make sure that themembrane of the purification column has been spin-dried, becauseresidual ethanol will interfere with subsequent reactions.

Putting the purification column in a new 1.5 mL or 2 mL centrifuge tube(provided by yourself), adding 200 μL of Buffer AE, dropping it directlyon the middle white membrane, placed at room temperature for 1 min, thencentrifuged at 6000 g (or 8000 rpm) for 1 min to elute DNA.

The eluted DNA was quantified with Nanodrop, and the sample was dilutedto a concentration of 10 ng/μL, and 10 μL was taken for STR detection.

(3) STR Detection

Selecting 16 STR locus for identification of identical twins family,including: CSF1PO, D135317, D165539, D18551, D195433, D21S11, D2S1338,D3S1358, D5S818, D7S820, D8S1179, FGA, THO1, TPDX, vWA, and designingthe corresponding primers for subsequent PCR verification.

PCR amplification: completing fluorescent PCR according to the designedexperimental scheme, and using agarose electrophoresis to detect theresults;

On-board detection: PCR products were detected by capillaryelectrophoresis using ABI 3730XL sequencing instrument to obtain data onthe size of amplified fragments. Using GeneMarker v1.9 to process andanalyze the collected data, converting the size and quantity of productfragments into intuitive and accurate waveform maps, and performinggenetic analysis to get the results of genetic relationship.

FIG. 2 shows the results of genetic relationship of Fudan_D5, Fudan_D6,Fudan_F7, Fudan_M8 identified by the STR method. The 16 STR locus suchas CSF1PO are all human genetic markers. Based on existing data and DNAanalysis results, it is supported that Fudan_F7 and Fudan_M8 are thebiological parents of Fudan_D5 and Fudan_D6, and Fudan_D5 and Fudan_D6are identical twins. In addition, the DNA of the cell line is consistentwith the identification results of DNA of the volunteer's original bloodsample.

Example 3 Comparison of DNA from Cell Lines and Original Blood Samples

The Illumina HiSeq XTen whole-genome sequencing platform was used toperform whole-genome sequencing on the DNA of volunteers' original bloodsamples and cell lines, and to evaluate the influence of theestablishment process on the whole genome sequence.

(1) Method of Library Constructing

The library was constructed using TruSeq Nano DNA Library Prep Kit,according to the method of TruSeq DNA Sample Preparation Guide(Illumina, 15026486 Rev. C).

(2) Sequence Determination

Using paired-end 150 bp sequencing, the average sequencing depth is 45×.The sequencing reagent adopts HiSeq XTen Reagent Kit v2.5.

(3) Bioinformatics Analysis

After the quality control of the original sequencing data, bwa-mem wasused for sequence alignment (mapping). The reference genome used washg19, and the alignment results were preprocessed by GATK (mainlyincluding eliminating the influence of excessive PCR and re-alignmentand correction of indel mutations, base correction, etc.) and variantcalling was performed by using HaplotypCaller. Two variant detectionmodes were used for each sample: conventional variant detection (VCFresults, only sites that were inconsistent with the reference genomewere reported) and gVCF results (all sites were reported). Then, theconsistency of the original blood sample and the whole genome site ofthe cell line was counted. Among them, the number of inconsistent siteswas counted using the VCF results of the original blood sample and cellline, and the number of detectable sites was counted using the gVCFresults of the original blood sample and cell line.

The results are shown in Table 1. The consistency between Fudan_D5,Fudan_D6, Fudan_F7, Fudan_M8 and the corresponding original blood samplewhole genome locus is 99.976%, 99.980%, 99.974%, 99.977%, respectively.

TABLE 1 Consistency results of the whole genome sequence of theimmortalized cell line and the original blood sample Number of sitesNumber of Consistency inconsistent detectable rate Cell line with bloodsample sites (%) Fudan_D5 87,505 359,719,460 99.976 Fudan_D6 87,445427,124,551 99.980 Fudan_F7 87,660 337,853,423 99.974 Fudan_M8 87,946386,291,050 99.977

Example 4 Effect Analysis of Quadruple Standard Product VS TripleStandard Product

In this example, in order to compare the detection effects of thequadruple standard product and the triple standard product, identicaltwin families were used as genomic reference substances to performMendelian genetic error analysis.

Methods were shown as below:

Using the Illumina HiSeq XTen whole-genome sequencing platform, wholegenome sequencing was performed for the DNA of the four cell lines. Thesequencing errors were evaluated according to the Mendelian geneticrules of the family, and comparing the advantages of the detection rateof sequencing errors in identical twins families over the detection rateof ordinary families of three.

(1) Method of Library Constructing

The library was constructed using TruSeq Nano DNA Library Prep Kit,according to the method of TruSeq DNA Sample Preparation Guide(Illumina, 15026486 Rev. C).

(2) Sequence Determination

Using paired-end 150 bp sequencing, the average sequencing depth is 45×.The sequencing reagent adopts HiSeq XTen Reagent Kit v2.5.

(3) Bioinformatics Analysis

After the quality control of the original sequencing data, bwa-mem wasused for sequence alignment (mapping). The reference genome used washg19, and the alignment results was preprocessed by GATK (mainlyincluding eliminating the influence of excessive PCR and re-alignmentand correction of indel mutations, base correction, etc.) and thevariant calling was performed by using HaplotypCaller. Two variantdetection modes were used for each sample: conventional variantdetection (VCF results, only sites that were inconsistent with thereference genome were reported) and gVCF results (all sites werereported). Then, the consistency of the original blood sample and thewhole genome site of the cell line was counted. Among them, the numberof inconsistent sites was counted using the VCF results of the originalblood sample and cell line, and the number of detectable sites wascounted using the gVCF results of the original blood sample and cellline.

The results are shown in Table 2. Trio1 is the Mendelian genetic errorrate calculated with Fudan_D5, Fudan_F7, Fudan_M8, Trio2 is theMendelian genetic error rate calculated with Fudan_D6, Fudan_F7,Fudan_M8, and the independently detected Mendelian genetic error ratesof the Trio1 and Trio2 families of the three technical duplicationfamilies are all between 0.33 and 0.35%. In addition to the Mendeliangenetic error rate of Trio1 and Trio2, the identical twin family(Quartet) can also detect inconsistent Fudan_D5 and Fudan_D6 genotypesloci, but the condition is that they meet the genetic rules of Trio1 andTrio2, respectively. A Mendelian genetic error rate of 0.9% can bedetected.

TABLE 2 Number of detectable sites Error rate(%) Total Fudan_D5 Fudan_D6Fudan_F7 Fudan_M8 Trio1 Trio2 Quartet Ti/Tv 1 6,503,734 4,840,9794,836,825 4,814,322 4,827,198 0.33 0.35 0.87 1.98 2 6,512,814 4,846,9264,846,390 4,822,709 4,843,043 0.36 0.36 0.92 1.98 3 6,512,489 4,839,3504,847,558 4,814,966 4,835,124 0.35 0.35 0.90 1.98

The results show that it can be seen from Table 4 that when the triplestandard products are used, their detection rate for Mendelianinheritance is only between 0.33 and 0.35%, and when the quadruplestandard products are used, they increase the detection rate ofMendelian inheritance by about 200%, which can reflect that the data canbe mutually verified by adding quadruple standard products, thereby moreeffectively detecting more Mendelian genetic errors. This suggests thatthe quadruple standard product of the present invention can provide moremutual verification information when evaluating the capabilities anddetection performance of the detection platform, so as to be able tomore comprehensively evaluate the performance indicators of thedetection platform, including: precision, accuracy, sensitivity,specificity, detectable range.

In addition, it can be seen from the above that a synergistic detectioneffect can be achieved more effectively.

Example 4 Evaluation on the Quality of Methylation Detection Chip byQuadruple Standard Products

In this embodiment, when identical twin families were used as referencesubstances for epigenome, transcriptome, proteome, and metabolome, anintrinsic magnitude difference gradient can be formed to evaluate thesensitivity of omics detection.

In this embodiment, DNA standard products were used to detectdifferences in methylation of identical twin families.

Methylation Detection:

Reference substances at the epigenome, transcriptome, proteome,metabolome and other levels of identical twin families, and theirintrinsic magnitude differences, that is, Fudan_D5 and Fudan_D6 weremore similar in terms of epigenome, transcriptome, proteome, metabolome,etc., and the magnitude difference was smaller than the differencebetween that and Fudan_F7 and Fudan_M8. This inherent difference inmagnitude of identical twins was innovative in evaluating thequantitative accuracy of technical platforms such as epigenome,transcriptome, proteome, and metabolome, etc.

FIG. 3 shows that the DNA samples of Fudan_D5, Fudan_D6, Fudan_F7, andFudan_M8 are tested using Illumina Methylation 850K chip for 3 technicalreplicates, respectively. The Heatmap results show that from themethylation level, Fudan_D5 and Fudan_D6 have the highest similarity,and followed by Fudan_F7, Fudan_M8. This result shows that the IlluminaMethylation 850K chip has high technical reproducibility and can welldetect differences in methylation of identical twins.

The results in FIG. 3 show that at the DNA methylation level, threetechnical replicates for each of the quadruple standard product canobtain consistent clustering results, that is, Fudan_D5 and Fudan_D6cluster first, followed by Fudan_F7 and Fudan_M8. This result shows thatthe quadruple standard product of the present invention has a stableintrinsic magnitude difference in DNA methylation level, and can be usedto evaluate the performance of a methylation detection platform,including: precision, accuracy, sensitivity, and specificity, detectablerange.

Example 5 Evaluation on RNA Transcriptome of Quadruple Standard Product

In this embodiment, the standard product of RNA was used to detect thedifference in the expression level of the identical twins families.

FIG. 4 is the use of Hiseq 4000, Ribo-zero library construction methodto perform 2 technical replicates detection of Fudan_D5, Fudan_D6,Fudan_F7, Fudan_M8 RNA samples, respectively. Heatmap results show thatFudan_D5, Fudan_D6 have the highest similarity, followed by Fudan_F7,Fudan_M8. This result shows that RNA-Seq has high technicalreproducibility and can well detect differences in the expression levelsof identical twins.

The results in FIG. 4 show that at the RNA expression level, twotechnical duplicates for each of the quadruple standard products canobtain consistent clustering results, that is, Fudan_D5 and Fudan_D6cluster first, followed by Fudan_F7 and Fudan_M8. The results indicatethat quadruple standard product of the present invention has stableinternal magnitude differences in RNA expression levels, and can be usedto evaluate the performance of the transcriptome detection platform,including: precision, accuracy, sensitivity, specificity, anddetectability range.

It can be seen from Examples 4, 5, and 6, that regardless of the DNAlevel, DNA methylation level, and RNA level, more mutually confirmed andricher information can be provided. This suggests that it is verysuitable as a standard product for a more comprehensive evaluation ontesting platform, including, but not limited to: sequencing platform,chip detection platform, metabolite detection platform, methylationdetection platform, transcriptome detection platform, proteome detectionplatform.

Culture Preservation

The human immortalized B lymphocyte cell line Fudan_D5 (the same as thedeposit name) of the present invention has been deposited in ChinaCenter for Type Culture Collection on Nov. 8, 2017, address: WuhanUniversity, Wuhan, China, deposit number: CCTCC NO: C2017238.

Culture Preservation

The human immortalized B lymphocyte cell line Fudan_D6 (the same as thedeposit name) of the present invention has been deposited in ChinaCenter for Type Culture Collection on Nov. 8, 2017, address: WuhanUniversity, Wuhan, China, deposit number: CCTCC NO: C2017253.

Culture Preservation

The human immortalized B lymphocyte cell line Fudan_F7 (the same as thedeposit name) of the present invention has been deposited in ChinaCenter for Type Culture Collection on Nov. 8, 2017, address: WuhanUniversity, Wuhan, China, deposit number: CCTCC NO: C2017254.

Culture Preservation

The human immortalized B lymphocyte cell line Fudan_M8 (the same as thedeposit name) of the present invention has been deposited in ChinaCenter for Type Culture Collection on Nov. 8, 2017, address: WuhanUniversity, Wuhan, China, deposit number: CCTCC NO: C2017255.

All literatures mentioned in the present application are incorporated byreference herein, as though individually incorporated by reference.Additionally, it should be understood that after reading the aboveteaching, many variations and modifications may be made by the skilledin the art, and these equivalents also fall within the scope as definedby the appended claims.

1. A cell line for constructing a standard product for judging theaccuracy of a detection platform, wherein the cell line is selected fromthe group consisting of: human immortalized B lymphocyte line Fudan_D5,human immortalized B Lymphocyte line Fudan_D6, human immortalized Blymphocyte line Fudan_F7, and/or human immortalized B lymphocyte lineFudan_M8; and, the deposit number of human immortalized B lymphocyteline Fudan_D5 is CCTCC NO: C2017238, the deposit number of the humanimmortalized B lymphocyte line Fudan_D6 is CCTCC NO: C2017253, thedeposit number of the human immortalized B lymphocyte line Fudan_F7 isCCTCC NO: C2017254, and the deposit number of the human immortalized Blymphocyte line Fudan_M8 is CCTCC NO: C2017255.
 2. The cell line ofclaim 1, wherein the cell line comprises human immortalized B lymphocyteline Fudan_D5, human immortalized B lymphocyte line Fudan_D6, humanimmortalized B lymphocyte line Fudan_F7 and human immortalized Blymphocyte line Fudan_M8.
 3. A standard product, which is extracted fromthe cell line of claim
 1. 4. The standard product of claim 3, whereinthe standard product comprises a set of standards, and each standard isderived from the cell line of claim 1, respectively.
 5. The standardproduct of claim 4, wherein the standard comprises a first standard, asecond standard, a third standard, and a fourth standard; wherein, thefirst standard is extracted from a human immortalized B lymphocyte lineFudan_D5, the second standard is extracted from human immortalized Blymphocyte line Fudan_D6, the third standard is extracted from humanimmortalized B lymphocyte line Fudan_F7, the fourth standard isextracted from human immortalized B lymphocyte line Fudan_M8.
 6. Use ofthe standard product of claim 3 for the preparation of a reagent and/orkit for judging the accuracy of a detection platform.
 7. The use ofclaim 6, wherein the detection platform is selected from the groupconsisting of: a sequencing platform, a chip detection platform, ametabolite detection platform, a methylation detection platform, atranscriptome detection platform, a proteome detection platform, and acombination thereof.
 8. A method for judging the accuracy of a detectionplatform to be tested, comprising the steps: (a) providing a standardproduct of claim 3 and a platform to be tested; (b) constructing alibrary for the standard product, thereby obtaining a sequencinglibrary; (c) sequencing the sequencing library of step (b) using theplatform to be tested, thereby obtaining a sequencing result; (d)comparing the sequencing result with the standard data (threshold value)of the standard sequence corresponding to the standard product, therebyobtaining the performance confirmation parameters of the method,including precision, accuracy, sensitivity, specificity, and/ordetectable range, thereby evaluating and verifying the accuracy of theplatform to be tested.
 9. The method of claim 8, wherein in step (d),comparing the sequencing result with the standard data (threshold value)of the standard sequence corresponding to the standard product, therebyobtaining the matching value of the sequencing result and the standarddata of the standard sequence, if the matching value is ≥99% (preferably≥99.5%, more preferably, 99.974-99.980%), indicating that the sequencingplatform is accurate or qualified; if the matching value is <98%,indicating that the sequencing platform is inaccurate or unqualified.10. A kit comprising: (a) a first container, and a first standardlocated in the first container, the first standard being extracted froma human immortalized B lymphocyte cell lineFudan_D5; (b) a secondcontainer, and a second standard located in the second container, thesecond standard being extracted from a human immortalized B lymphocytecell lineFudan_D6; (c) a third container, and a third standard locatedin the third container, the third standard being extracted from a humanimmortalized B lymphocyte cell lineFudan_F7; and (d) a fourth container,and a fourth standard located in the fourth container, the fourthstandard being extracted from a human immortalized B lymphocyte celllineFudan_M8.